Since the structure of DNA was deciphered by Watson & Crick in 1953 (Watson, J. D. and Crick, F. H. C., Nature 171 (1953) 737-738) investigation and handling of nucleic acids becomes an integral part of biochemistry molecular biology. Despite the availability of a number of isolation methods an commercial kits for performing such methods, new developments for fast and easy isolation or purification of nucleic acids with high yield and purity are still of major importance.
Nucleic acids are highly susceptible to enzymatic degradation. In 1968 Cox described the chaotropic agent guanidine HCl as an inhibitor of enzymatic nuclease activity (Cox, R. A., Methods Enzymol 12B (1968) 120-129). Besides a strong denaturing effect on proteins high concentrations of chaotropic agents also mediate cell lysis. Therefore chaotropic agents, particularly guanidine thiocyanate, are widely in use for nucleic acid isolation.
A first principle of nucleic acid isolation from a biological sample uses an organic solvent, particularly phenol, for the separation of nucleic acids from the remaining organic sample components. The phenol extraction is followed by a salt precipitation of the nucleic acid from an aqueous phase (Stallcup, M. R. and Washington, L. D., J. Biol. Chem. 258 (1983) 2802-2807, and Schmitt, M. E., et al., Nucl. Acid Res. 18 (1990) 3091-3092). Although this method results in nucleic acids with high yield and purity the major drawbacks are the use of poisonous reagents, the time consuming and labor intensive workflow. Due to these disadvantages automation of this isolation principle is not amenable to automation, or only to a very limited extent.
Another principle of nucleic acid isolation makes use of solid inorganic material, particularly silica, to which nucleic acids are adsorbed from an aqueous liquid phase such as a lysate of a biological sample. In 1979 Vogelstein and Gillespie described a method for isolating nucleic acid from agarose gel slices by binding nucleic acids to silica particles in presence of highly concentrated sodium iodide (Vogelstein, B. and Gillespie, D., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619).
In addition it was found that the binding of nucleic acids to the solid phase was increased by the addition of anionic or cationic or neutral detergents, in particular TRITON-X100 (Union Carbide Chemicals & Plastics Technology Corporation), sodium dodecyl sulfate, NP40, and TWEEN 20 (ICI Americas Inc.).
Adsorption of a nucleic acid to the solid phase is usually performed in the presence of a potent denaturant such as a chaotropic agent (Boom, R. et al., J. Clin. Microbiol. 28 (1990) 495-503; U.S. Pat. No. 5,234,809). For the isolation process the biological material is mixed with a solution containing the denaturant. The resulting mix is brought into contact with the solid phase material whereby nucleic acid molecules are bound to the surface of the solid phase. Afterwards the solid material is washed with solutions containing decreasing chaotropic salt concentrations and increasing alcohol concentrations, in particular ethanol, in order to further purify the bound nucleic acids from other organic material and contaminating agents. In the last step the solid material is brought into contact with a low salt solution or water under alkaline pH in order to remove the bound nucleic acid from the solid phase. The complete workflow comprises a sample lysis step, a binding step, one or more washing steps, and an elution (desorption) step.
The solid phase can be arranged in different conformations. In a first design the solid phase is in fleece shape and embedded in a plastic device. An example therefor is a micro spin column (EP 0 738 733). This design is preferentially used in workflows which are performed manually. In a second design magnetic silica particles are used as a solid phase (Bartl, K., et al., Clin. Chem. Lab. Med. 36 (1998) 557-559). This design is preferentially used in automated workflows.
A further improvement of this method was observed when aliphatic alcohol (i.e., ethanol or isopropanol) or polyethylene glycol is added to the solution at the binding step (U.S. Pat. No. 6,383,393).
US 2002/10192667 A1 discloses purification of nucleic acids by addition (in buffer) of a chaotrope and an organic solvent such as the cyclic ether compound 1,4-dioxane, after which the mixture is passed by an inorganic support such as silica, onto which the nucleic acids bind. Other examples of added organic solvents in the binding buffer include the aliphatic ethers ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran, the aliphatic esters propylene glycol monomethyl ether acetate, and ethyl lactate, and the aliphatic ketones hydroxyacetone, acetone, and methyl ethyl ketone.
US 2005/0079535 discloses the use of acetone, acetylacetone, acetonitrile, dimethylsulfoxide, diethylketone, methylethylketone, methylpropylketone, isobutylmethylketone, gamma-butyrolactone, gamma-valerolactone, propylene carbonate, and N-methyl-2-pyrrolidone as well as the use of the cyclic diether dioxane in the binding buffer, in order to adsorb a nucleic acid to a solid phase such as silica.
The methods for the isolation/purification of nucleic acids of the state of the art have certain disadvantages. Such disadvantages relate to, e.g., purity, selectivity, recovery rate, laboratory safety and convenience, as well as to the speed of the isolation/purification process.
E.g., in protocols using a phenol/chloroform extraction, residual phenol is often a problem for certain post isolation procedures, particularly for enzymatic reactions such as a digestion with a restriction enzyme, the polymerase chain reaction (PCR), or a ligase-mediated reaction. Generally, elevated concentrations of residual reagents from the purification/isolation process may pose a problem. It is therefore desired to keep residual amounts of the reagents used during the purification procedure as low as possible in the purified nucleic acid. Another potential problem related to purity is the coextraction of certain substances from the adsorption matrix (leaching). It is therefore desired to keep residual amounts of compounds liberated during the purification procedure by leaching as low as possible in the purified nucleic acid.
The chemical properties of the reagents used in the isolation/purification process determines the quality of the nucleic (yield, purity and size) as well as their performance in down-stream workflows, including polymerase or reverse transcriptase based enzymatic reactions (Mullis, K. and Faloona, F. A., Methods Enzymol 155 (1987) 335-350). Furthermore, additional properties of the reagents like toxicity, as well as physical and chemical aspects like flash point and vapor pressure are of major importance.
Hazardous substances often bear environmental risks and cause high costs for waste management. Their use can be restricted based on the required technical and/or operational safety measures to be taken. The hazardous potential of buffers used in the isolation/purification of nucleic acids is chiefly influenced by the choice of the organic compound which promotes adsorption of the nucleic acid to the solid phase. With respect to the environmental burden it is desired to reduce the use of toxic or harmful agents as far as possible. Also, the flash point of a flammable organic compound, that is the lowest temperature at which it can form an ignitable mixture with oxygen, is desired to be high. This parameter particularly influences the costs of production of kits for nucleic acid purification. An organic compound with a flash point below room temperature has to be handled in specially equipped production facilities which prevent the development of explosive vapour. In addition, restrictions apply to the transport of such organic compounds. A low flash point is usually correlated with a high vapor pressure. As a consequence, certain organic compounds, particularly lower alcohols, tend to evaporate from solutions and therefore lead to variations in concentration over time. This effect also influences stability during storage as well as the handling of liquids with a high vapor pressure in an automated pipetting instrument. Avoiding substances with low flash points and a tendency to evaporate in the isolation/purification process would make the production of solutions for the purification of nucleic acids simpler and more economical. In addition, compounds with a low vapour pressure are desired as they increase the utility of nucleic acid isolation kits by eliminating a major source of pipetting error, thereby increasing the reliability of such kits.
The problem underlying the present invention therefore was to provide alternative compounds to promote the adsorption of a nucleic acid to a solid phase.
The inventors have surprisingly found that adsorption of a nucleic acid to a solid phase is effectively accomplished when a water-miscible liquid cyclic acetal is used in the adsorption solution.